1. Field of the Invention
This invention relates to an analysis element for measuring a photographically active substance to be measured or a compound labelled with a photographically active substance which is employed for a method of measuring a trace amount of a compound labelled with the photographically active substance by the use of the photographically active substance and silver halide in combination, and to a method for immunochemical measurement of a trace component using the same.
2. Development of the Invention
As methods for measuring a trace component utilizing the combination of a photographically active substance and silver halide, the following methods can be exemplified: for example, methods in which a spectral sensitizer is employed as the photographically active substance--which have been previously proposed by the present inventors--are as follows:
(I) A method for immunologically measuring trace components which comprises:
competitively reacting an antigen or antibody labelled with a spectral sensitizer and an antigen or antibody to be measured with an antibody or antigen which specifically reacts with the antigen or antibody, PA1 bringing either the thus formed dye-labelled antigen-antibody reaction product or the unreacted antigen or antibody into contact with silver halide, PA1 exposing the same to light having a spectrally sensitizing wavelength corresponding to the spectral sensitizer, PA1 developing the exposed silver halide, and, PA1 measuring the resulting optical density of the developed silver or colored dye; PA1 using a synthetic substrate containing a substrate structure to be specifically contacted with an enzyme to be measured and labelled with a spectral sensitizer, PA1 bringing either the reaction product containing the spectral sensitizer formed by enzyme reaction between the synthetic substrate and the enzyme to be measured or the unreacted synthetic substrate into contact with silver halide, PA1 exposing the same to light having a spectrally sensitizing wavelength of the spectral sensitizer, and, PA1 measuring the resulting optical density of the developed silver or colored dye; PA1 competitively reacting an antigen or antibody labelled with a spectral sensitizer and a testing sample containing an antigen or antibody to be measured with an antibody or antigen which specifically reacts with the respective antigen species or antibody species, PA1 bringing either the thus formed reaction product of the unreacted matter into contact with a silver halide light sensitive material, PA1 exposing the same to light having a wavelength which the spectral sensitizer absorbs, PA1 developing the exposed silver, and, PA1 quantitatively measuring the antigen or antibody based upon the optical density of the resulting silver image or the color density obtained. PA1 using a synthetic substrate containing at least one spectral sensitizer structure for photographic use, i.e., an organic dye structure 2 which has an absorption region at a longer wavelength (preferably, longer than 500 nm) than the absorption wavelength inherent to silver halide and which spectrally sensitizes silver halide grains by contact with (adsorption to) the silver halide grains and at least one structure 1 to be specifically contacted with the enzyme to be measured, PA1 bringing either the reaction product containing the spectral sensitizer structure 2 formed by the enzyme reaction or the unreacted synthetic substrate with a silver halide light-sensitive material, PA1 exposing the same to light at a wavelength region which the spectral sensitizer sensitizes, PA1 developing, and, PA1 measuring the quantity of the developed silver or the colored dye as optical density. PA1 using a synthetic substrate containing at least one structure 1 to be specifically contacted with an enzyme to be measured and at least one spectral sensitizer structure or a fogging agent structure 2 in the same molecule thereof, PA1 bringing either the reaction product containing the spectral sensitizer structure or fogging agent structure formed by enzyme reaction between the synthetic substrate and the enzyme to be measured or the unreacted synthetic substrate in contact with silver halide, PA1 exposing the same to light having a wavelength which the corresponding spectral sensitizer used absorbs, followed by development, when the synthetic substrate containing the spectral sensitizer is used, and when the synthetic substrate containing the fogging atent is used, developing the same without performing exposure, and, PA1 measuring the quantity of the enzyme from the blackened density or/and colored dye density. PA1 R.sub.3 and R.sub.4 have the same meanings as R and R.sub.1 ; R.sub.5 has the same meaning as R.sub.2 ; r has the same meaning as n; L.sub.1 and L.sub.2 are as defined above; PA1 m.sub.1 represents 2, 3 or 4; PA1 d represents 1, 2 or 3; PA1 Q.sub.1 represents an oxygen atom, a sulfur atom or --N--R.sub.6 (R.sub.6 represents an aliphatic group); PA1 Q represents a non-metallic atomic group necessary to complete a 5-membered or 6-membered nitrogen-containing heterocyclic nucleus. PA1 L.sub.1 to L.sub.5 are the same as L.sub.1 to L.sub.3 ; PA1 R.sub.10 is the same as R.sub.4 ; Q.sub.2 is the same as Q.sub.1 ; PA1 k and l represent 1, 2 or 3, and may be the same or different. PA1 (1) Spectral sensitizers are directly reacted with the aforesaid functional groups; PA1 (2) Spectral sensitizers and the aforesaid functional groups are reacted using an activator, and PA1 (3) Spectral sensitizers and the aforesaid functional groups are reacted via at least one compound having a bifunctional group. PA1 1. Compounds containing a cyclic or acyclic thiocarbonyl group (e.g., thioureas, dithiocarbamates, trithiocarbonates, dithioesters, thioamides, rhodanines, thiohytandoins, thiosemicarbazides, or derivatives thereof) PA1 2. Compounds containing a cyclic or acyclic thio ether group (e.g., sulfides, disulfides, polysulfides, etc.) PA1 3. Other sulfur-containing compounds (e.g., thiosulfates, thiophosphates, and compounds derived therefrom PA1 4. Nitrogen-containing reducible compounds (e.g., hydrazines, hydrazones, amines, polyamines, cyclic amines, hydroxylamines, quaternary ammonium salt derivatives, etc.) PA1 5. Reducible compounds (e.g., aldehydes, sulfinic acids, enediols, metal hydride compounds, alkyl metals, aromatic compounds in dihydro form, active methylene compounds, etc.) PA1 6. Metal complexes (e.g., four-coordinate Ni(II) or Fe(II) complexes having sulfur as a ligand, etc.) PA1 7. Acetylene compounds PA1 8. Others (phosphonium salts, etc.) PA1 e.g., a compound of the following formula: ##STR9## and aldehyde compounds as described in Japanese Patent Application OPI No. 9678/72, Japanese Patent Publication Nos. 19452/77, and 20088/74, etc. PA1 e.g., metal hydride compounds as described in Japanese Patent Publication No. 28065/70, U.S. Pat. Nos. 3,951,665 and 3,804,632, British Pat. No. 821,251, etc. PA1 (1) Straight, branched and cyclic alkyl groups, preferably containing 1 to 20 carbon atoms, such as a methyl group, an ethyl group, an isopropyl group, an n-dodecyl group, and a cyclohexyl group; PA1 (2) Aralkyl groups, preferably monocyclic and bicyclic aralkyl groups having an alkyl moiety containing 1 to 3 carbon atoms, such as a benzyl group; PA1 (3) Alkoxy groups, preferably containing 1 to 20 carbon atoms, such as a methoxy group and an ethoxy group; PA1 (4) Amino groups, preferably an --NH.sub.2 group and those amino groups mono- or di-substituted by an alkyl group containing 1 to 20 carbon atoms, such as a dimethylamino group and a diethylamino group; PA1 (5) Aryloxy groups, preferably a phenoxy group; PA1 (6) Groups represented by Ah--Xh--(Y).sub.nh -- PA1 (7) Groups represented by ##STR17## (8) Groups represented by R.sup.3h CONHNH--Ar--Y"--. PA1 (a) Xh is a divalent linking group selected from the following x.sub.1 to x.sub.11 : x.sub.1 =--CSNH--, x.sub.2 =--S--CSNH--, ##STR18## x.sub.4 =--CONH--, x.sub.5 =--O--E--CONH--, ##STR19## x.sub.7 =--NHCO--, x.sub.8 =--O--, x.sub.9 =--SO.sub.2 NH--, x.sub.10 =--E--NH--, and x.sub.11 =--E.dbd.N--; PA1 (b) Y is a divalent linking group selected from the following y.sub.1 to y.sub.11 : y.sub.1 =--CONH--, y.sub.2 =--E--CONH--, y.sub.3 =--E--, y.sub.4 =--E--O--E'--, y.sub.5 =--E--S--E'--, y.sub.6 =--SO.sub.2 NH--, y.sub.7 =--E--SO.sub.2 NH--, y.sub.8 =--NHCONH--, y.sub.9 =--E--NHCONH--, y.sub.10 =--E--O--E'--CONH--, and y.sub.11 =--E--E'--, PA1 R.sub.11 is a hydrogen atom, an aliphatic group (preferably, an alkyl group containing 1 to 20 carbon atoms, a cycloalkyl group containing 3 to 12 carbon atoms, or an alkenyl group containing 2 to 20 carbon atoms), or an aromatic group (preferably, a phenyl group and a naphthyl group), PA1 R.sub.12 is a hydrogen atom or an aliphatic group represented by R.sub.11, PA1 R.sub.11 and R.sub.12 may combine with each other to form a ring, with preferred examples of such ring being ##STR20## (in this case, Ah represents hydrogen), when R.sub.11 and R.sub.12 do not form a ring, any one of R.sub.11 and R.sub.12 is a hydrogen atom, and PA1 E and E' each represents a saturated or unsaturated divalent aliphatic group (e.g., an alkylene group, such as an ethylene group and a 1-methylpropylene group, and an alkenylene group, such as a propenylene group and a butenylene group), a divalent aromatic group (e.g., a phenylene group, a naphthylene group and a 5-amino-1,2-phenylene group), with the exception that in y.sub.11 =--E--E'--, E and E' are divalent groups different from each other and in x.sub.11 =--E.dbd.N--, E is --(CH.sub.2).sub.mh --CH.dbd. (wherein m is an integer of 0 to 2); PA1 (c) nh is an integer of 0 or 1, and when nh is 1, particularly preferred combinations of Xh and Y are x.sub.3 -y.sub.2, x.sub.7 -y.sub.2, x.sub.8 -y.sub.2, x.sub.12 -y.sub.3, x.sub.3 -y.sub.7, x.sub.5 -y.sub.9, x.sub.9 -y.sub.9, and x.sub.3 -y.sub.10 ; and PA1 (d) Ah represents a straight, branched or cyclic alkyl group (preferably containing 1 to 20 carbon atoms, such as a methyl group, a propyl group, and an n-hexyl group), a monocyclic or bicyclic aryl group (e.g., a phenyl group), a monocyclic or bicyclic aralkyl group (preferably containing 7 to 26 carbon atoms, such as a benzyl group), and a heterocyclic radical. PA1 an alkoxy group (preferably containing 1 to 18 carbon atoms, such as a methoxy group), PA1 an alkoxycarbonyl group (preferably containing 2 to 19 carbon atoms, such as an ethoxycarbonyl group), PA1 a monocyclic or bicyclic aryl group (e.g., a phenyl group), PA1 an alkyl group (preferably containing 1 to 20 carbon atoms, such as a methyl group and a tert-amyl group), PA1 a dialkylamino group (preferably containing 1 to 20 carbon atoms, such as a dimethylamino group), PA1 an alkylthio group (preferably containing 1 to 20 carbon atoms, such as a methylthio group), PA1 a mercapto group, a hydroxy group, a halogen atom, a carboxy group, a nitro group, a cyano group, PA1 a sulfonyl group (preferably containing 1 to 20 carbon atoms, such as a methylsulfonyl group), and PA1 a carbamoyl group (preferably containing 1 to 20 carbon atoms, such as a carbamoyl group and a dimethylcarbamoyl group). PA1 (b) Bh is a hydrogen atom or a saturated or unsaturated aliphatic group [such as an alkyl group (preferably containing 1 to 20 carbon atoms, e.g., a methyl group and an ethyl group), an alkenyl group (preferably containing 2 to 22 carbon atoms, e.g., an allyl group), and an alkynyl group (preferably containing 2 to 20 carbon atoms, e.g., a butynyl group)], which may be substituted by an alkoxy group, an alkylthio group, an acylamino group, an acyloxy group, a mercapto group, a sulfo group, a carboxy group, a hydroxy group, a halogen atom, an amino group, or the like; PA1 (c) Y' has the same meanings as described for Y in Group (6); and PA1 (d) nh is 0 or 1. PA1 (a) R.sup.3h is the same as R.sup.2 as described hereinafter; PA1 (c) Y" is the same as Y described in Group (6), with divalent linking groups represented by y.sub.3 to y.sub.5 being particularly preferred.
(II) A method for quantitatively measuring a trace enzyme which comprises:
(III) A method for detecting the location or distribution of a corresponding antibody or antigen or its receptor in tissue, utilizing the same dye labelled antigen or antibody as used in (I) above, in combination with silver halide.
In the case where the photographically active substance is a fogging agent, trace components can be measured in the same manner as above, except that the exposure procedure is omitted.
As a method for measuring trace components utilizing specificity in the antigen-antibody reaction shown in (I) and (III), radioimmunoassay (RIA) is known. The basic principles of RIA corresponding to, e.g., (I), are as foolows.
That is, the reaction of a substance marked (labelled) with a radioactive isotope (RI) in a given amount and a substance having a specific binding affinity thereto in a given amount results in a bound product of both of these components, while a part of the labelled substance remains in an unbound or unreacted free state. The reaction proceeds based on the laws of mass action in general, and, therefore, when an unlabelled substance is added to the reaction system, binding with a limited amount of binding protein is decreased and a certain relationship (calibration curve) can be established therebetween. As a result, an amount of an unknown substance can be determined from the calibration curve if the bound substance and the labelled substance in the free state are separated and either one or both are measured with respect to the RI amount.
Due to the high sensitivity and the simplicity of RIA, RIA is particularly applicable to the measurement and inspection of trace amounts of proteins in blood and hormones. Details thereon are given in, e.g., Kumahara and Shizume, NEW RADIOIMMUNOASSAY, pages 3 to 10 (1977), published by Asakura Publishing Co., Ltd., KISO SEIKAGAKU JIKKENHO (Basic Biochemical Experiment), (6), Biochemical Assay (1967), published by Maruzen Co., Ltd.
However, RIA is subject to several disadvantages due to the use of RI labelling substances (.sup.125 I, .sup.131 I, etc.) which must have high specific radioactivity to maintain immune activity and must be of high purity. For these reasons, RIA involves the danger of radiation exposure and it is necessary to use expensive and unstable labelling substances which cannot be used for extended periods of time. In addition, special installations, equipment and personnel qualified to deal with radiation are required. Finally, after RIA, disposal of radioactive waste material and the ensuing pollution problems are encountered.
Other methods for measuring the activity of an enzyme as a trace component are also known.
For example, there are: a turbidimetry method which comprises tracing the decrease in turbidity due to enzyme reaction using a suspension of a high molecular weight substrate; an absorptiometric method which comprises decomposing a high molecular weight substrate, precipitating undecomposed substrate, recovering it and then measuring soluble matter by an absorbance measurement; a method for quantitative measurement which comprises binding a dye or fluorescent substance to a high molecular weight substrate, effecting enzyme reaction to decrease the molecular weight of the dye or fluorescent substance, and measuring the dye or fluorescent substance separated; a method of quantitative measurement which comprises using a substrate which is designed to change in absorption spectrum, form a color or form a fluorescent substance, based on a splitting-off or change in a part of the substrate after enzyme reaction, and measuring the resulting absorbancy or fluorescent intensity, etc. (SEIKAGAKU JIKKEN KOZA (Lectures on Biochemical Experiments), vol. 5, Study on Enzymes, edited by the Biochemical Association, Japan, published by Tokyo Kagaku Dojin, 1979).
Most of these methods quantitatively determine an amount of enzyme on the order of .mu.g/ml. Even utilizing a type of substrate releasing a fluorescent substance (e.g., derivatives of coumarine, umbelliferone, etc.), which is recognized to be most sensitive among these methods, it is only possible to measure an enzyme quantity on the order of ng/ml.
The importance of quantitative measurement of enzyme traces in blood or body fluids, the distribution of the enzymes in the living body, the amount excreted in urine, etc., has increased, and RIA has begun to be put in practice in the areas where one cannot measure activity according to the other methods above described. In addition to the problems described above, the RIA method has the following shortcomings:
(1) There is the possibility that the activity which is a functional characteristic of an enzyme is not correctly reflected because of immunoassay.
(2) There is the possibility that analogous enzymes and precursors having a similar antigenic site might be included in the analytical data.
(3) In the case where an enzyme to be measured, for example, an enzyme in an antigen or antibody labelled with the enzyme used for enzyme immunoassay, is conjugated with another compound and is not present in free form, it is difficult to prepare an antibody and design for a measurement method is practically difficult.
For these reasons, it has been desired to develop a method for the immunological examination of trace components or measurement of enzyme activity which is stable and provides sufficient sensitivity without using any isotope.
The inventors have discovered a method for the measurement of trace components with high sensitivity applicable to immunological examination and enzyme immunoassay using a photographically active substance, e.g., a spectral sensitizer, a fogging agent, etc., in combination with silver halide. Details of immunological examination are described in U.S. Ser. Nos. 126,920 and 126,919, both filed Mar. 3, 1980.
The invention will now be described with reference to the case wherein a spectral sensitizer is employed as the photographically active substance.
When a trace component to be measured as in (I) described above is, e.g., an antigen or antibody, the method is practiced as follows.
That is, the immunochemical measurement method comprises:
Further, in the case where a trace component to be measured as in (II) described above is an enzyme, the method is practiced as follows.
That is, the method comprises, in the measurement of enzyme activity;
Method (II) above has also been proposed by the present inventors (see copending application Ser. No. 298,814 filed Sept. 2, 1981).
The term "synthetic substrate" used herein refer to a substrate synthesized in the laboroatory as opposed to substrates derived from living tissues and is recognized in the art (see Japanese Patent Application OPI 52691/77). In the syntehtic substrate, structure 1 generally comprises a site to be catalytically affected by an enzyme to be measured (in other words, a site to be catalytically, e.g., cleaved, with the enzyme) and a site to be specifically recognized by the enzyme (i.e., a recognition site or binding site) and thus specifically contacted with the enzyme to be measured.
The enzymatic reaction which occurs in Method (II) of this invention can be illustrated as shown below.
______________________________________ ##STR1## ##STR2## .circle.1 : structure 1.circle. .circle.2 : structure 2.circle. .circle.1' : functionally changed from 1.circle.on contact with enzyme .circle.1 unreacted synthetic substance .circle.1' 2.circle.: reaction product of enzymatic reaction : linkage directly linking 1.circle.and 2.circle.or indirectly via linking group 3.circle.
More specifically, structure which is present in the synthetic substrate used in the method of the present invention and specifically contacted with an enzyme to be measured is generally composed of a site to be contacted with the enzyme such as a peptide bond (acid amide bond), an ester bond, a phosphate bond, a glucoside bond for, e.g., hydrolase; or an amino group, a carboxy group, etc. for, e.g., transferase; and a site to be recognized by the enzyme (binding site) such as an amino acid residue, sugar, a nucleic acid base, etc. These are described with regard to respective enzymes described later in more detail as substrate structures corresponding to substrate specifities of enzymes in DATA BOOK OF BIOCHEMISTRY, first and second separate volumes (1979 and 1980), edited by the Biochemical Association, Japan, published by Tokyo Kagaku Dojin and Paul. D., Boyer et al. The Enzyme, vols. I, III, IV and V, 1971, published by Academic Press.
The synthetic substrate used in this invention refers to a substrate comprising linking thereto at least one structure 1 corresponding to the aforesaid substrate specificity and at least one spectral sensitizer structure 2 which will be later explained. Requirements for the linking are that 1 enzyme reactivity not be inhibited and 2 spectral sensitizing capability not be lost by the linking.
In addition to (I) and (II) described above, the method of the present invention is also applicable to the determination of the distribution state of components in the living body in tissue, such as receptor assay using the labelled antigen or antibody (receptor assay using, e.g., radioisotopes is described in detail in RADIOIMMUNOASSAY, Second series, edited by Minoru Irie, Chapter 12, published by Kodansha Publishing Co., Ltd.) and can be utilized for the quantitative measurement of various components in the living body, drugs, trace components such as enzymes or the like.
For measurement of trace components, an analysis sheet conventionally used, i.e., an analysis sheet comprising a support and a light sensitive silver halide emulsion layer (hereafter referred to as an "emulsion layer") has been employed in the prior art. However, contact of a trace component(s) labelled with a photographically active substance with silver halide--required in the present invention--was not sufficient with the prior art analysis sheet having such a characteristic feature. Therefore, it is necessary to enhance detection sensitivity, reproducibility, etc., in order to achieve highly sensitive measurement of trace components.